Project Summary The receptor activator of NF-?B ligand (RANKL) was initially identified as an essential factor regulating osteoclast formation and function. RANKL exerts its function by activating its cognate receptor RANK (the receptor activator of NF-?B). Given the crucial role of RANKL in osteoclast biology, Amgen has successfully developed a humanized anti-RANKL antibody (denosumab) as a new drug for osteoporosis. In addition, subsequent studies demonstrated that RANKL also plays a role in mammary gland development. Importantly, RANKL has been further shown to play a role in progesterone-induced and Brca1 mutation-driven breast cancer development. These findings suggest that the blockade of the RANKL function may be a novel therapeutic strategy for preventing and treating breast cancer development. However, denosumab causes various side effects including osteonecrosis of jaw and increased risk for serious infections since RANKL regulates a variety of other biological processes including the immune function. We previously identified three functional TRAF (TNF receptor associated factor)-binding motifs in the RANK cytoplasmic domain (Motif 1: PFQEP369-373; Motif 2: PVQEET559-564; Motif 3: PVQEQG604-609) that regulate osteoclast formation. Intriguingly, Motif 1 activates all of the known RANK signaling pathways (NF-?B, JNK, p38, ERK and Akt pathways) but it exerts minimal effects on osteoclast formation. In contrast, while Motifs 2 and 3 activate only one or two of the known RANK signaling pathways, they are very potent in promoting osteoclast formation. In particular, dual mutation of Motifs 2 and 3 dramatically impairs RANK signaling in osteoclast formation in vitro. To assess the role of Motifs 2 and 3 in osteoclast formation in vivo, we have successfully generated knockin mice (KI) bearing inactivating mutations in RANK Motifs 2 and 3. Osteoclast formation is dramatically reduced in the KI mice, confirming the role of these two RANK motifs in osteoclast formation in vivo. Interestingly, we have also found that mammary gland development is impaired in the KI mice. This finding suggests that these two RANK motifs primarily mediate the RANK's function in mammary gland development. More importantly, this finding has also raised a possibility that these two RANK motifs may also be involved in progesterone-induced and Brca1 mutation-driven breast cancer development. Moreover, we found that the dual mutation of RANK Motifs 2 and 3 does not affect the capacity of RANK to activate NF-?B, MAPK and Akt pathways, which regulate immune cell development and function. Based on these data, we hypothesize that RANK Motifs 2 and 3 have the potential to serve as effective and selective drug targets for preventing and treating breast cancer. The objective of this R21 proposal is to carry out crucial in vitro and mouse model studies to address this hypothesis. Significantly, validation of these two RANK motifs as effective and selective drug targets for breast cancer will facilitate future development of efficacious and safe drugs for preventing and treating breast cancer.